Tectorigenin protects against cardiac fibrosis in diabetic mice heart via activating the adiponectin receptor 1-mediated AMPK pathway.

Diabetic cardiomyopathy (DCM) is a common severe complication of diabetes that occurs independently of hypertension, coronary artery disease, and valvular cardiomyopathy, eventually leading to heart failure. Previous studies have reported that Tectorigenin (TEC) possesses extensive anti-inflammatory and anti-oxidative stress properties. In this present study, the impact of TEC on diabetic cardiomyopathy was examined. The model of DCM in mice was established with the combination of a high-fat diet and STZ treatment. Remarkably, TEC treatment significantly attenuated cardiac fibrosis and improved cardiac dysfunction. Concurrently, TEC was also found to mitigate hyperglycemia and hyperlipidemia in the DCM mouse. At the molecular level, TEC is involved in the activation of AMPK, both in vitro and in vivo, by enhancing its phosphorylation. This is achieved through the regulation of endothelial-mesenchymal transition via the AMPK/TGFß/Smad3 pathway. Furthermore, it was demonstrated that the level of ubiquitination of the adiponectin receptor 1 (AdipoR1) protein is associated with TEC-mediated improvement of cardiac dysfunction in DCM mice. Notably the substantial reduction of myocardial fibrosis. In conclusion, TEC improves cardiac fibrosis in DCM mice by modulating the AdipoR1/AMPK signaling pathway. These findings suggest that TEC could be an effective therapeutic agent for the treatment of diabetic cardiomyopathy.

The two-component system expression patterns and immune regulatory mechanism of Vibrio parahaemolyticus with different genotypes at the early stage of infection in THP-1 cells.

Vibrio parahaemolyticus must endure various challenging circumstances while being swallowed by phagocytes of the innate immune system. Moreover, bacteria should recognize and react to environmental signals quickly in host cells. Two-component system (TCS) is an important way for bacteria to perceive external environmental signals and transmit them to the interior to trigger the associated regulatory mechanism. However, the regulatory function of V. parahaemolyticus TCS in innate immune cells is unclear. Here, the expression patterns of TCS in V. parahaemolyticus-infected THP-1 cell-derived macrophages at the early stage were studied for the first time. Based on protein-protein interaction network analysis, we mined and analyzed seven critical TCS genes with excellent research value in the V. parahaemolyticus regulating macrophages, as shown below. VP1503, VP1502, VPA0021, and VPA0182 could regulate the ATP-binding-cassette (ABC) transport system. VP1735, uvrY, and peuR might interact with thermostable hemolysin proteins, DNA cleavage-related proteins, and TonB-dependent siderophore enterobactin receptor, respectively, which may assist V. parahaemolyticus in infected macrophages. Subsequently, the potential immune escape pathways of V. parahaemolyticus regulating macrophages were explored by RNA-seq. The results showed that V. parahaemolyticus might infect macrophages by controlling apoptosis, actin cytoskeleton, and cytokines. In addition, we found that the TCS (peuS/R) could enhance the toxicity of V. parahaemolyticus to macrophages and might contribute to the activation of macrophage apoptosis. IMPORTANCE This study could offer crucial new insights into the pathogenicity of V. parahaemolyticus without tdh and trh genes. In addition, we also provided a novel direction of inquiry into the pathogenic mechanism of V. parahaemolyticus and suggested several TCS key genes that may assist V. parahaemolyticus in innate immune regulation and interaction.

Cdo1 promotes PPARγ-mediated adipose tissue lipolysis in male mice.

Cysteine dioxygenase 1 (Cdo1) is a key enzyme in taurine synthesis. Here we show that Cdo1 promotes lipolysis in adipose tissue. Adipose-specific knockout of Cdo1 in mice impairs energy expenditure, cold tolerance and lipolysis, exacerbates diet-induced obesity (DIO) and decreases adipose expression of the key lipolytic genes encoding ATGL and HSL, with little effect on adipose taurine levels. White-adipose-specific overexpression of ATGL and HSL blunts the role of adipose Cdo1 deficiency in promoting DIO. Mechanistically, Cdo1 interacts with PPARγ and facilitates the recruitment of Med24, the core subunit of mediator complex, to ATGL and HSL gene promoters, thereby transactivating their expression. Further, mice with transgenic overexpression of Cdo1 show better cold tolerance, ameliorated DIO and higher lipolysis capacity. Thus, we uncover an unexpected and important role of Cdo1 in regulating adipose lipolysis.

Breast MRI Tumor Automatic Segmentation and Triple-Negative Breast Cancer Discrimination Algorithm Based on Deep Learning.

Background: Breast cancer is a kind of cancer that starts in the epithelial tissue of the breast. Breast cancer has been on the rise in recent years, with a younger generation developing the disease. Magnetic resonance imaging (MRI) plays an important role in breast tumor detection and treatment planning in today's clinical practice. As manual segmentation grows more time-consuming and the observed topic becomes more diversified, automated segmentation becomes more appealing. Methodology. For MRI breast tumor segmentation, we propose a CNN-SVM network. The labels from the trained convolutional neural network are output using a support vector machine in this technique. During the testing phase, the convolutional neural network's labeled output, as well as the test grayscale picture, is passed to the SVM classifier for accurate segmentation. Results: We tested on the collected breast tumor dataset and found that our proposed combined CNN-SVM network achieved 0.93, 0.95, and 0.92 on DSC coefficient, PPV, and sensitivity index, respectively. We also compare with the segmentation frameworks of other papers, and the comparison results prove that our CNN-SVM network performs better and can accurately segment breast tumors. Conclusion: Our proposed CNN-SVM combined network achieves good segmentation results on the breast tumor dataset. The method can adapt to the differences in breast tumors and segment breast tumors accurately and efficiently. It is of great significance for identifying triple-negative breast cancer in the future.

Limonin stabilises sirtuin 6 (SIRT6) by activating ubiquitin specific peptidase 10 (USP10) in cardiac hypertrophy.

BACKGROUND AND PURPOSE: Limonin, a naturally occurring tetracyclic triterpenoid, has extensive pharmacological effects. Its role in cardiac hypertrophy remains to be elucidated. We investigated its effects on cardiac hypertrophy along with the potential mechanisms involved. EXPERIMENTAL APPROACH: The effects of limonin on cardiac hypertrophy in C57/BL6 mice caused by aortic banding, plus neonatal rat cardiac myocytes (NRCMs) stimulated with phenylephrine to induce cardiomyocyte hypertrophy in vitro were investigated. KEY RESULTS: Limonin markedly improved the cardiac function and heart weight in aortic banded mice. Limonin-treated mice and NRCMs also produced fewer cardiac hypertrophy markers than those treated with the vehicle in the hypertrophic groups. Sustained aortic banding- or phenylephrine-stimulation impaired cardiac sirtuin 6 (SIRT6) protein levels, which were partially reversed by limonin associated with enhanced activity of PPARα. Sirt6 siRNA inhibited the anti-hypertrophic effects of limonin in vitro. Interestingly, limonin did not influence Sirt6 mRNA levels, but regulated ubiquitin levels. Thus, the protein biosynthesis inhibitor, cycloheximide and proteasome inhibitor, MG-132, were used to determine SIRT6 protein expression levels. Under phenylephrine stimulation, limonin increased SIRT6 protein levels in the presence of cycloheximide, but it did not influence SIRT6 expression in the presence of MG-132, suggesting that limonin promotes SIRT6 levels by inhibiting its ubiquitination degradation. Furthermore, limonin inhibited the degradation of SIRT6 by activating ubiquitin-specific peptidase 10 (USP10), while Usp10 siRNA prevented the beneficial effects of limonin. CONCLUSION AND IMPLICATIONS: Limonin mediates the ubiquitination and degradation of SIRT6 by activating USP10, providing an attractive therapeutic target for cardiac hypertrophy.

Screening of Lipid Metabolism-Related Gene Diagnostic Signature for Patients With Dilated Cardiomyopathy.

Background: Dilated cardiomyopathy (DCM) is characterized by enlarged ventricular dimensions and systolic dysfunction and poor prognosis. Myocardial lipid metabolism appears abnormal in DCM. However, the mechanism of lipid metabolism disorders in DCM remains unclear. Methods: A gene set variation analysis (GSVA) were performed to estimate pathway activity related to DCM progression. Three datasets and clinical data downloaded from the Gene Expression Omnibus (GEO), including dilated cardiomyopathy and donor hearts, were integrated to obtain gene expression profiles and identify differentially expressed genes related to lipid metabolism. GO enrichment analyses of differentially expressed lipid metabolism-related genes (DELs) were performed. The clinical information used in this study were obtained from GSE21610 dataset. Data from the EGAS00001003263 were used for external validation and our hospital samples were also tested the expression levels of these genes through RT-PCR. Subsequently, logistic regression model with the LASSO method for DCM prediction was established basing on the 7 DELs. Results: GSVA analysis showed that the fatty acid metabolism was closely related to DCM progression. The integrated dataset identified 19 DELs, including 8 up-regulated and 11 down-regulated genes. A total of 7 DELs were identified by further external validation of the data from the EGAS00001003263 and verified by RT-PCR. By using the LASSO model, 6 genes, including CYP2J2, FGF1, ETNPPL, PLIN2, LPCAT3, and DGKG, were identified to construct a logistic regression model. The area under curve (AUC) values over 0.8 suggested the good performance of the model. Conclusion: Integrated bioinformatic analysis of gene expression in DCM and the effective logistic regression model construct in our study may contribute to the early diagnosis and prevention of DCM in people with high risk of the disease.

SERPINA3C ameliorates adipose tissue inflammation through the Cathepsin G/Integrin/AKT pathway.

OBJECTIVE: Due to the increasing prevalence of obesity and insulin resistance, there is an urgent need for better treatment of obesity and its related metabolic disorders. This study aimed to elucidate the role of SERPINA3C, an adipocyte secreted protein, in obesity and related metabolic disorders. METHODS: Male wild type (WT) and knockout (KO) mice were fed with high-fat diet (HFD) for 16 weeks, adiposity, insulin resistance, and inflammation were assessed. AAV-mediated overexpression of SERPINA3C was injected locally in inguinal white adipose tissue (iWAT) to examine the effect of SERPINA3C. In vitro analyses were conducted in 3T3-L1 adipocytes to explore the molecular pathways underlying the function of SERPINA3C. RESULTS: Functional exploration of the SERPINA3C knockout mice revealed that SERPINA3C deficiency led to an impaired metabolic phenotype (more severe obesity, lower metabolic rates, worse glucose intolerance and insulin insensitivity), which was associated with anabatic inflammation and apoptosis of white adipose tissues. Consistent with these results, overexpression of SERPINA3C in inguinal adipose tissue protected mice against diet-induced obesity and metabolic disorders with less inflammation and apoptosis in adipose tissue. Mechanistically, SERPINA3C inhibited Cathepsin G activity, acting as a serine protease inhibitor, which blocked Cathepsin G-mediated turnover of α5/ß1 Integrin protein. Then, the preserved integrity (increase) of α5/ß1 Integrin signaling activated AKT to decrease JNK phosphorylation, thereby inhibiting inflammation and promoting insulin sensitivity in adipocytes. CONCLUSIONS/INTERPRETATION: These findings demonstrate a previously unknown SERPINA3C/Cathepsin G/Integrin/AKT pathway in regulating adipose tissue inflammation, and suggest the therapeutic potential of targeting SERPINA3C/Cathepsin G axis in adipose tissue for the treatment of obesity and metabolic diseases.

Lupeol protects against cardiac hypertrophy via TLR4-PI3K-Akt-NF-κB pathways.

Inflammation and apoptosis are main pathological processes that lead to the development of cardiac hypertrophy. Lupeol, a natural triterpenoid, has shown anti-inflammatory and anti-apoptotic activities as well as potential protective effects on cardiovascular diseases. In this study we investigated whether lupeol attenuated cardiac hypertrophy and fibrosis induced by pressure overload in vivo and in vitro, and explored the underlying mechanisms. Cardiac hypertrophy was induced in mice by transverse aortic constriction (TAC) surgery, and in neonatal rat cardiomyocytes (NRCMs) by stimulation with phenylephrine (PE) in vitro. We showed that administration of lupeol (50 mg ·kg-1· d-1, i.g., for 4 weeks) prevented the morphological changes and cardiac dysfunction and remodeling in TAC mice, and treatment with lupeol (50 µg/mL) significantly attenuated the hypertrophy of PE-stimulated NRCMs, and blunted the upregulated hypertrophic markers ANP, BNP, and ß-MHC. Furthermore, lupeol treatment attenuated the apoptotic and inflammatory responses in the heart tissue. We revealed that lupeol attenuated the inflammatory responses including the reduction of inflammatory cytokines and inhibition of NF-κB p65 nuclear translocation, which was mediated by the TLR4-PI3K-Akt signaling. Administration of a PI3K/Akt agonist 740 Y-P reversed the protective effects of lupeol in TAC mice as well as in PE-stimulated NRCMs. Moreover, pre-treatment with a TLR4 agonist RS 09 abolished the protective effects of lupeol and restored the inhibition of PI3K-Akt-NF-κB signaling by lupeol in PE-stimulated NRCMs. Collectively, our results demonstrate that the lupeol protects against cardiac hypertrophy via anti-inflammatory mechanisms, which results from inhibiting the TLR4-PI3K-Akt-NF-κB signaling.

Mouse strain-specific responses of mitochondrial respiratory function and cardiac hypertrophy to isoproterenol treatment.

Cardiac hypertrophy is a common pathological process of various cardiovascular diseases and eventually develops into heart failure. This paper was aimed to study the different pathological characteristics exhibited by different mouse strains after hypertrophy stimulation. Two mouse strains, A/J and FVB/nJ, were treated with isoproterenol (ISO) by osmotic pump to induce cardiac hypertrophy. Echocardiography was performed to monitor heart morphology and function. Mitochondria were isolated from hearts in each group, and oxidative phosphorylation function was assayed in vitro. The results showed that both strains showed a compensatory enhancement of heart contractile function after 1-week ISO treatment. The A/J mice, but not the FVB/nJ mice, developed significant cardiac hypertrophy after 3-week ISO treatment as evidenced by increases in left ventricular posterior wall thickness, heart weight/body weight ratio, cross sectional area of cardiomyocytes and cardiac hypertrophic markers. Interestingly, the heart from A/J mice contained higher mitochondrial DNA copy number compared with that from FVB/nJ mice. Functionally, the mitochondria from A/J mice displayed faster O2 consumption at state III with either complex I substrates or complex II substrate, compared with those from FVB/nJ mice. ISO treatment did not affect mitochondrial respiratory control rate (RCR), but significantly suppressed the ADP/O ratio generated from the complex II substrate in both strains. The ADP/O ratio generated from the complex I substrates in A/J mice declined by 50% after ISO treatment, whereas FVB/nJ mice were not affected. These results suggest that, compared with FVB/nJ mice, A/J mice possesses a poor integrity of mitochondrial respiratory chain that might contribute to its vulnerability to ISO-induced cardiac hypertrophy.

l-Theanine Activates the Browning of White Adipose Tissue Through the AMPK/α-Ketoglutarate/Prdm16 Axis and Ameliorates Diet-Induced Obesity in Mice.

l-Theanine is a nonprotein amino acid with much beneficial efficacy. We found that intraperitoneal treatment of the mice with l-theanine (100 mg/kg/day) enhanced adaptive thermogenesis and induced the browning of inguinal white adipose tissue (iWAT) with elevated expression of Prdm16, Ucp1, and other thermogenic genes. Meanwhile, administration of the mice with l-theanine increased energy expenditure. In vitro studies indicated that l-theanine induced the development of brown-like features in adipocytes. The shRNA-mediated depletion of Prdm16 blunted the role of l-theanine in promoting the brown-like phenotypes in adipocytes and in the iWAT of mice. l-theanine treatment enhanced AMPKα phosphorylation both in adipocytes and iWAT. Knockdown of AMPKα abolished l-theanine-induced upregulation of Prdm16 and adipocyte browning. l-Theanine increased the α-ketoglutarate (α-KG) level in adipocytes, which may increase the transcription of Prdm16 by inducing active DNA demethylation on its promoter. AMPK activation was required for l-theanine-induced increase of α-KG and DNA demethylation on the Prdm16 promoter. Moreover, intraperitoneal administration with l-theanine ameliorated obesity, improved glucose tolerance and insulin sensitivity, and reduced plasma triglyceride, total cholesterol, and free fatty acids in the high-fat diet-fed mice. Our results suggest a potential role of l-theanine in combating diet-induced obesity in mice, which may involve l-theanine-induced browning of WAT.

Mzb1 protects against myocardial infarction injury in mice via modulating mitochondrial function and alleviating inflammation.

Myocardial infarction (MI) leads to the loss of cardiomyocytes, left ventricle dilation and cardiac dysfunction, eventually developing into heart failure. Mzb1 (Marginal zone B and B1 cell specific protein 1) is a B-cell-specific and endoplasmic reticulum-localized protein. Mzb1 is an inflammation-associated factor that participates a series of inflammatory processes, including chronic periodontitis and several cancers. In this study we investigated the role of Mzb1 in experimental models of MI. MI was induced in mice by ligation of the left descending anterior coronary artery, and in neonatal mouse ventricular cardiomyocytes (NMVCs) by H2O2 treatment in vitro. We showed that Mzb1 expression was markedly reduced in the border zone of the infarct myocardium of MI mice and in H2O2-treated NMVCs. In H2O2-treated cardiomyocytes, knockdown of Mzb1 decreased mitochondrial membrane potential, impaired mitochondrial function and promoted apoptosis. On contrary, overexpression of Mzb1 improved mitochondrial membrane potential, ATP levels and mitochondrial oxygen consumption rate (OCR), and inhibited apoptosis. Direct injection of lentiviral vector carrying Len-Mzb1 into the myocardial tissue significantly improved cardiac function and alleviated apoptosis in MI mice. We showed that Mzb1 overexpression significantly decreased the levels of Bax/Bcl-2 and cytochrome c and improved mitochondrial function in MI mice via activating the AMPK-PGC1α pathway. In addition, we demonstrated that Mzb1 recruited the macrophages and alleviated inflammation in MI mice. We conclude that Mzb1 is a crucial regulator of cardiomyocytes after MI by improving mitochondrial function and reducing inflammatory signaling pathways, implying a promising therapeutic target in ischemic cardiomyopathy.

The Effects of Maternal Obesity on Porcine Placental Efficiency and Proteome.

Maternal obesity is associated with impaired maternal metabolism and affects the developmental programming of the fetus. The placenta is dysfunctional when exposed to an obese intrauterine environment and can transduce and mediate detrimental maternal impacts to the fetus through mechanisms that remain largely unknown. The main objective of this study was to investigate the effects of maternal obesity on the porcine placental proteome and to analyze the deregulated proteins and potential pathways predicted to be disturbed in obese placentas, using sows with high backfat as a model of obese pregnancy. The sows were divided into two groups based on their backfat thickness: normal backfat (NBF, 17-22 mm; n = 30) and high backfat (HBF, ≥23 mm; n = 30) as the maternal obesity group. The placental tissues used for the proteomic and biochemical analyses were obtained through vaginal delivery, and the maternal blood samples used to determine the metabolic parameters were collected at day 107 of pregnancy. Our study demonstrated that HBF sows had significantly decreased placental efficiency, increased plasma-free fatty acids and triglyceride levels, and increased proinflammatory cytokines plasma levels (p < 0.05). HBF placentas had significantly higher malondialdehyde level, lower total antioxidant capacity and antioxidase activity, increased triglyceride content and enhanced proinflammatory tumor necrosis factor- α (TNF-α) and interleukin-6 (IL-6) contents (p < 0.05). Among the 4652 proteins identified using the proteomic method, 343 were quantified as differentially abundant proteins, which were involved in many vital biological processes. Based on our bioinformatic and placental biochemical analyses, we concluded that maternal obesity is associated with abnormal carbohydrate and lipid metabolism, mitochondrial dysfunction, decreased steroid hormone biosynthesis, and increased oxidative stress and inflammation in the placenta. The results of this study are undoubtedly valuable to other researchers.

Taurine-mediated browning of white adipose tissue is involved in its anti-obesity effect in mice.

Taurine, a nonprotein amino acid, is widely distributed in almost all animal tissues. Ingestion of taurine helps to improve obesity and its related metabolic disorders. However, the molecular mechanism underlying the protective role of taurine against obesity is not completely understood. In this study, it was found that intraperitoneal treatment of mice with taurine alleviated high-fat diet (HFD)-induced obesity, improved insulin sensitivity, and increased energy expenditure and adaptive thermogenesis of the mice. Meanwhile, administration of the mice with taurine markedly induced the browning of inguinal white adipose tissue (iWAT) with significantly elevated expression of PGC1α, UCP1, and other thermogenic genes in iWAT. In vitro studies indicated that taurine also induced the development of brown-like adipocytes in C3H10T1/2 white adipocytes. Knockdown of PGC1α blunted the role of taurine in promoting the brown-like adipocyte phenotypes in C3H10T1/2 cells. Moreover, taurine treatment enhanced AMPK phosphorylation in vitro and in vivo, and knockdown of AMPKα1 prevented taurine-mediated induction of PGC1α in C3H10T1/2 cells. Consistently, specific knockdown of PGC1α in iWAT of the HFD-fed mice inhibited taurine-induced browning of iWAT, with the role of taurine in the enhancement of adaptive thermogenesis, the prevention of obesity, and the improvement of insulin sensitivity being partially impaired. These results reveal a functional role of taurine in facilitating the browning of white adipose tissue, which depends on the induction of PGC1α. Our studies also suggest a potential mechanism for the protective role of taurine against obesity, which involves taurine-mediated browning of white adipose tissue.

Enhanced acetylation of ATP-citrate lyase promotes the progression of nonalcoholic fatty liver disease.

Hepatic steatosis is a hallmark of nonalcoholic fatty liver disease (NAFLD) and is promoted by dysregulated de novo lipogenesis. ATP-citrate lyase (ACLY) is a crucial lipogenic enzyme that is up-regulated in individuals with NAFLD. A previous study has shown that acetylation of ACLY at Lys-540, Lys-546, and Lys-554 (ACLY-3K) increases ACLY protein stability by antagonizing its ubiquitylation, thereby promoting lipid synthesis and cell proliferation in lung cancer cells. But the functional importance of this regulatory mechanism in other cellular or tissue contexts or under other pathophysiological conditions awaits further investigation. Here, we show that ACLY-3K acetylation also promotes ACLY protein stability in AML12 cells, a mouse hepatocyte cell line, and found that the deacetylase sirtuin 2 (SIRT2) deacetylates ACLY-3K and destabilizes ACLY in these cells. Of note, the livers of mice and humans with NAFLD had increased ACLY protein and ACLY-3K acetylation levels and decreased SIRT2 protein levels. Mimicking ACLY-3K acetylation by replacing the three lysines with three glutamines (ACLY-3KQ variant) promoted lipid accumulation both in high glucose-treated AML12 cells and in the livers of high-fat/high-sucrose (HF/HS) diet-fed mice. Moreover, overexpressing SIRT2 in AML12 cells inhibited lipid accumulation, which was more efficiently reversed by overexpressing the ACLY-3KQ variant than by overexpressing WT ACLY. Additionally, hepatic SIRT2 overexpression decreased ACLY-3K acetylation and its protein level and alleviated hepatic steatosis in HF/HS diet-fed mice. Our findings reveal a posttranscriptional mechanism underlying the up-regulation of hepatic ACLY in NAFLD and suggest that the SIRT2/ACLY axis is involved in NAFLD progression.

Histone demethylase KDM5A is transactivated by the transcription factor C/EBPß and promotes preadipocyte differentiation by inhibiting Wnt/ß-catenin signaling.

ß-Catenin signaling is triggered by WNT proteins and is an important pathway that negatively regulates adipogenesis. However, the mechanisms controlling the expression of WNT proteins during adipogenesis remain incompletely understood. Lysine demethylase 5A (KDM5A) is a histone demethylase that removes trimethyl (me3) marks from lysine 4 of histone 3 (H3K4) and serves as a general transcriptional corepressor. Here, using the murine 3T3-L1 preadipocyte differentiation model and an array of biochemical approaches, including ChIP, immunoprecipitation, RT-qPCR, and immunoblotting assays, we show that Kdm5a is a target gene of CCAAT/enhancer-binding protein ß (C/EBPß), an important early transcription factor required for adipogenesis. We found that C/EBPß binds to the Kdm5a gene promoter and transactivates its expression. We also found that siRNA-mediated KDM5A down-regulation inhibits 3T3-L1 preadipocyte differentiation. The KDM5A knockdown significantly up-regulates the negative regulator of adipogenesis Wnt6, having increased levels of the H3K4me3 mark on its promoter. We further observed that WNT6 knockdown significantly rescues adipogenesis inhibited by the KDM5A knockdown. Moreover, we noted that C/EBPß negatively regulates Wnt6 expression by binding to the Wnt6 gene promoter and repressing Wnt6 transcription. Further experiments indicated that KDM5A interacts with C/EBPß and that their interaction cooperatively inhibits Wnt6 transcription. Of note, C/EBPß knockdown impaired the recruitment of KDM5A to the Wnt6 promoter, which had higher H3K4me3 levels. Our results suggest a mechanism involving C/EBPß and KDM5A activities that down-regulates the Wnt/ß-catenin pathway during 3T3-L1 preadipocyte differentiation.

Krüppel-like factor 10 (KLF10) is transactivated by the transcription factor C/EBPß and involved in early 3T3-L1 preadipocyte differentiation.

Adipose tissue stores energy and plays an important role in energy homeostasis. CCAAT/enhancer-binding protein ß (C/EBPß) is an important early transcription factor for 3T3-L1 preadipocyte differentiation, facilitating mitotic clonal expansion (MCE) and transactivating C/EBPα and peroxisome proliferator-activated receptor-γ (PPARγ) to promote adipogenesis. C/EBPß is induced early, but the expression of antimitotic C/EBPα and PPARγ is not induced until ∼48 h. The delayed expression of C/EBPα and PPARγ is thought to ensure MCE progression, but the molecular mechanism for this delay remains elusive. Here, we show that the zinc-finger transcription factor Krüppel-like factor 10 (KLF10) is induced after adipogenic induction and that its expression positively correlates with that of C/EBPß but inversely correlates with expression of C/EBPα and PPARγ. C/EBPß bound to the KLF10 promoter and transactivated its expression during MCE. KLF10 overexpression in 3T3-L1 preadipocyte repressed adipogenesis and decreased C/EBPα and PPARγ expression, whereas siRNA-mediated down-regulation of KLF10 enhanced adipogenesis and increased C/EBPα and PPARγ expression. Luciferase assays revealed an inhibitory effect of KLF10 on C/EBPα promoter activity. Using promoter deletion and mutation analysis, we identified a KLF10-binding site within the proximal promoter region of C/EBPα. Furthermore, KLF10 interacted with and recruited histone deacetylase 1 (HDAC1) to the C/EBPα promoter, decreasing acetylated histone H4 on the C/EBPα promoter and inactivating C/EBPα transcription. Because C/EBPα can transactivate PPARγ, our results suggest a mechanism by which expression of C/EBPα and PPARγ is delayed via KLF10 expression and shed light on the negative feedback loop for C/EBPß-regulated adipogenesis in 3T3-L1 preadipocyte.

Knockdown of ANRIL aggravates H2O2-induced injury in PC-12 cells by targeting microRNA-125a.

Spinal cord injury (SCI) is a devastating and common neurological disorder which causes local oxidative damage. The study aimed to investigate the underlying role of ANRIL in H2O2-induced cell injury of rat PC-12 cells. Cell injury was evaluated on the basis of cell viability, migration, invasion and apoptosis. The effect of ANRIL on H2O2-induced cell injury was estimated after cell transfection. Then, the interaction between ANRIL and miR-125a was explored by qRT-PCR and estimation of cell injury. Predicted by TargetScan, the possible target gene of miR-125a was verified. After that, the effects of aberrantly expressed target gene on cell viability, migration, invasion and apoptosis as well as phosphorylation of key kinases involved in JAK/STAT and ERK/MAPK pathways were evaluated. Results revealed that H2O2-induced PC-12 cell injury could be aggravated by ANRIL suppression. ANRIL appeared to act as a sponge of miR-125a, and ANRIL suppression promoted H2O2-induced cell injury by up-regulation of miR-125a. MCL-1 was a target of miR-125a, and MCL-1 was negatively correlated with miR-125a. Subsequent experiments showed the effect of MCL-1 silence on H2O2-induced PC-12 cell injury was the same as ANIRL suppression. MCL-1 attenuated H2O2-induced PC-12 cell injury by activating JAK/STAT and ERK/MAPK pathways. These findings suggested that knockdown of ANRIL aggravates H2O2-induced injury in PC-12 cells by targeting miR-125a. This might provide novel insights in the role of ANRIL in pathogenesis of oxidative damage during SCI.

Microbial Synthesis of 5-Aminolevulinic Acid and Its Coproduction with Polyhydroxybutyrate.

5-Aminolevulinic acid (ALA), an important cell metabolic intermediate useful for cancer treatments or plant growth regulator, was produced by recombinant Escherichia coli expressing the codon optimized mitochondrial 5-aminolevulinic acid synthase (EC: 2.3.1.37, hem1) from Saccharomyces cerevisiae controlled via the plasmid encoding T7 expression system with a T7 RNA polymerase. When a more efficient autoinduced expression approach free of IPTG was applied, the recombinant containing antibiotic-free stabilized plasmid was able to produce 3.6 g/L extracellular ALA in shake flask studies under optimized temperature. A recombinant E. coli expressing synthesis pathways of poly-3-hydroxybutyrate (PHB) and ALA resulted in coproduction of 43% PHB in the cell dry weights and 1.6 g/L extracellular ALA, leading to further reduction on ALA cost as two products were harvested both intracellularly and extracellularly. This was the first study on coproduction of extracellular ALA and intracellular PHB for improving bioprocessing efficiency. The cost of ALA production could be further reduced by employing a Halomonas spp. TD01 able to grow and produce ALA and PHB under continuous and unsterile conditions even though ALA had the highest titer of only 0.7 g/L at the present time.

[Effect of strains and parts on amino acids of Dendrobium officinale].

The aim of the paper is to reveals the variations of Dendrobium officinale amino acids in different strains and parts for breeding excellent varieties, and providing scientific basis for the expanding of medicinal or edible parts. The contents of 17 amino acids in 11 strains of D. officinale were determined by hydrochloric acid hydrolysis method. The total amino acids content of leaves was from 6.76 to 7.97 g per 100 g, and the stems was from 1.61 to 2.44 g per 100 g. As the content of amino acids in leaves was significantly higher than that of stems, and the composition was close to the ideal protein standard proposed by FAO/WHO. The leaves of D. officinale had the good prospect for the development of functional foods. The 9 x 66 strain which with high yield and polysaccharide content had the highest amino acids content both in stems and leaves, indicated crossbreeding could improve the quality of varieties. Compared the amino acids content of D. officinale in two main harvest periods, the harvest time has a significant impact on amino acids content of D. officinale. This study demonstrates that the harvesting time of D. officinale stems is suitable for leaves as well, which is the period before bolssom.